BACKGROUND: In tandem mass spectrometry, analyte detection is based on collision-induced fragmentation, which is modulated by the collision energy (CE) setting. Variation in CE leads to differential ion yield, and optimization is usually performed empirically as "tuning" during method development. Our aim was to build a method to objectify the impact of collision energy settings on ion yield for individual compounds. METHODS: Collision energy (CE)-breakdown curves were generated based on acquisition files in which a large number of quasi-identical mass transitions were recorded simultaneously, with variation of CE over a defined range within a single injection. Ion yield (normalized to an internal standard recorded with a locked collision energy) was plotted as a curve versus CE settings. Piperacillin and testosterone were studied as exemplary analytes in matrix-free and serum matrix-based liquid chromatography tandem mass spectrometry (LC-MS/MS) measurements. More detailed testosterone CE-breakdown curves were investigated with regard to sample preparation techniques and the isotope labeling pattern of corresponding internal standards. RESULTS: CE-breakdown curves were found characteristically for the piperacillin quantifier transition with respect to CE-related maximum ion yield, as well as curve width and shape. A diverging curve profile was observed for the piperacillin qualifier transition. For testosterone analyses, no impact from different sample preparation techniques or the isotope labeling patterns on the selected CE was shown. CONCLUSION: CE-breakdown curves are a convenient and valuable tool to verify LC-MS/MS methods regarding consistent fragmentation characteristics between sample sources or native analytes and isotope-labeled counterparts.
BACKGROUND: In tandem mass spectrometry, analyte detection is based on collision-induced fragmentation, which is modulated by the collision energy (CE) setting. Variation in CE leads to differential ion yield, and optimization is usually performed empirically as "tuning" during method development. Our aim was to build a method to objectify the impact of collision energy settings on ion yield for individual compounds. METHODS: Collision energy (CE)-breakdown curves were generated based on acquisition files in which a large number of quasi-identical mass transitions were recorded simultaneously, with variation of CE over a defined range within a single injection. Ion yield (normalized to an internal standard recorded with a locked collision energy) was plotted as a curve versus CE settings. Piperacillin and testosterone were studied as exemplary analytes in matrix-free and serum matrix-based liquid chromatography tandem mass spectrometry (LC-MS/MS) measurements. More detailed testosterone CE-breakdown curves were investigated with regard to sample preparation techniques and the isotope labeling pattern of corresponding internal standards. RESULTS: CE-breakdown curves were found characteristically for the piperacillin quantifier transition with respect to CE-related maximum ion yield, as well as curve width and shape. A diverging curve profile was observed for the piperacillin qualifier transition. For testosterone analyses, no impact from different sample preparation techniques or the isotope labeling patterns on the selected CE was shown. CONCLUSION: CE-breakdown curves are a convenient and valuable tool to verify LC-MS/MS methods regarding consistent fragmentation characteristics between sample sources or native analytes and isotope-labeled counterparts.